The present invention relates to a combined vaccine, its production and use, in particularly to hepatitis A-measles combined vaccine formulations comprising prophylactically effective titers of hepatitis A virus (HAV) and measles virus (MV) as virus components, and stabilizer components which is effective for stabilizing the virus components. The combined vaccine does not induce any interference on the immune response and the immunogenicity of the two viruses and having excellent storage stability, so that the combined vaccine could be stored at ambient temperature for a long time and could be used for preventing recipients, especially intants and young children against the two infections diseases. The present invention further relates to methods of making the combined vaccines which are lyophilized or unlyophilized by mixing the two different viruses or by propagating the two different viruses on a same diploid cell substrate.
As well known that, immunization to protect against communicable disease is one of the most successful and cost-effective practices of modern medicine. In order to total control over the entire vaccination chain leading to increased social acceptance and use of vaccine in all countries and regions, especially in developing countries and regions, the measures including providing newer, polyvalent, more stable and cost-effective vaccines must be taken. For this purpose, so much research efforts for developing newer vaccines and vaccines having improved stability has been extensively made in many laboratories of the world, since the xe2x80x9cChildren""s Vaccine Initiativexe2x80x9d (CVI) had founded by United Nations Children""s Fund (UNCF) and World Health Organization (WHO) in 1991. Therein, much attention is focused on making vaccine more user-friendly through development of combined vaccine and the introduction of less invasive inoculation technigues.
Popularization of the combined vaccines reduced the nember and manufacturing cost of vaccines, and increased immunity levels against disease, so that it has greatly increased vaccine coverage in the world, especially in developing countries including China.
Many combined vaccine have been licensed and used in immunization practices, for example in the past several decades a routine vaccination schedule for infants and children has included immunization with a live attenuated trivalent vaccine (MMR) for measles, mupms and rubella, and live trivalent vaccine (DTP) for diphtheria, tetanus and pertussis and so on. Recently, a haemophilus b conjugate vaccine combined by reconstitution with diphtheria and tetanus toxoids and acellular pertussis vaccine (DTaP-Hib) for use as fourth dose in childhood vaccination series has licenced by Food and Drug Administration (FDA) in the United States. Also, TriHlBit (Trademark) will be the first vaccine to be licensed combined vaccine in U.S. that combines PTaP with a haemophilus b conjugate vaccine. However, there has been no reports on combined vaccine comprising hepatitis A and measles viruses and on processes of making combined vaccine by propagating two or more different viruses on a same cells substrates.
The present invention provides a combined hepatitis A-measles vaccine (HM) comprising prophylactically effective titers of attenuated hepatitis A live virus and prophylactically effective titers of attenuated measles live virus, and with or without stabilizer for the viruses, by means of which vaccine the infection associated with hepatitis A virus and measles virus can be prevented without causing any interference between antigenicity of the two viruses due to their mixing or co-culturing.
The combined hepatitis A-measles vaccine according to the present invention, wherein the viral titers of hepatitis A virus is not less than 106.0 CCID50/ml (50% cell culture infectious dose per milliliter of virus stock), and the viral titers of measles virus is not less than 103.5 CCLD50/ml.
Within the scope of the present invention, the combined hapatitis A-measles vaccine is provided in the form of lyophilized or unlyophilized.
The present invention further provides a stabilized combined hepatitis A-measles vaccine formulation comprising virus components and stabilizer components, wherein the virus components comprise prophylactically effective titers of hepatitis A live virus and measles live virus, and the stabilizer components consisting essentially of from 0 to 2% (w/v) of human serum albumin (HSA), from 0.5 to 1.1% (w/v) of gelatin, from 5 to 10%(w/v) of trehalose, from 0.75 to 1.5% (w/v) of sodium glutamate, from 0.05 to 0.5% (w/v) of ascorbic acid, from 0.5 to 2.8% (w/v) of urea, from 5 to 10% (w/v) of mannitol or sorbitol or a 1:1 mixture of them, and from 0.5 to 1% (w/v) of nositol in terms of the concentrations in the stabilized live vaccine formulation prior to lyophilization, and wherein the stabilized combined vaccine optionally lyophilized or unlyophilized.
According to the present invention, there is provided a method of preparing combined hepatitis A-measles vaccine comprising mixing a stock material of attenuated hepatitis A live virus having viral titers of not less than 107.0 CCID50/ml with a stock material of attenuated measles live vaccine having viral titers of not less than 104.5 CCID50/ml at a suitable ratio, to thereby obtain a combined vaccine stock suspension, in which the viral titers of said hepatitis A virus is not less than 106.5 CCID50/ml and the viral titers of said measles virus is not less than 104.0 CCID50/ml.
The combined vaccine thus prepared is a useful vaccine which can be used to prevent the infections with both hepatitis A and measles without any reduction in immunogenic potencies and any deterioration of its properties. Moreover, the combined vaccine never caused interference between viral antigenecities due to their mixing, which interference is frequently abserved, in particulr in case of combined vaccine for animals.
According to the present invention, the present inventors are able to provide another method of preparing the combined hepatitis A-measles vaccine, comprising following steps:
(a) providing seed viruses of hepatitis A virus and measles virus previously prepared, respectively;
(b) inoculating the seed virus of hepatitis A virus obtained from step (a) into a human fetal lung diploid fibroblast cell cultures and following by culturing the cells to propagate the virus;
(c) when the positive hepatitis A virus-infected cells are more than 75%, inoculating the seed virus of measles virus obtained from step (a) into the same cell monolayers on which the hepatitis A virus have propagated, and the cells are cultured continuously;
(d) At the time when the positive hepatitis A virus-infected cells are more than 90% as detected in terms of immunofluoresence (IF) and the positive measles virus-infected cells are more than 90% as detected in terms of cytopathogenic effect (CPE), the cells infected with the two viruses are harvested and the vaccine stock materials containing the two viruses are collected to thereby obtain the desired combined vaccife.
In a preferred embodiment of the present invention, wherein the positive infectious rates on the diploid cells and viral titers of hepatitis A virus are detected by HAV-specific detection systems comprising immunofluorescence and enzyme-linked immunosorbent assay.
In a more preferred embodiment of the present invention, the method defined as above furthermore comprising suitable mixing the stock material of combined hepatitis A-measles vaccine obtained above with a stabilizer for attenuated live vaccine at about 1:1 volume ratio and then lyophilizing the resultant formulation thereby to obtain a lyophilized preparation of the combined hepatitis A-measles vaccine.
According to a preferred embodiment of the present invention, said stabilizer consisting essentially of from 0 to 2% (w/v) of human serum albumin, from 0.5 to 1% (w/v) of gelatin, from 5 to 10% (w/v) of trehalose, from 0.75 to 1.5% (w/v) of sodium glutamate, from 0.05 to 0.55% (w/v) of ascorbic acid, from 0.5 to 2.8% (w/v) of urea, from 5 to 10% (w/v) of mannitol or sorbitol or a 1:1 mixture of them, and from 0.5 to 1% (w/v) of inositol, in terms of the concentrations in the lyophilized live vaccine formulation.
The present invention still further provides a method of preparing lyophilized combined hepatitis A-measles vaccine, comprising following steps:
(a) providing stock materials comprising prophylactically effective titers of attenuated hepatitis A live virus and of attenuated measles live virus respectively, and then mixing them to obtain a mixed vaccine stock;
(b) adding a stabilizer stock solution to the mixed vaccine stock of step (a) at about 1:1 (v/v) ratio and mixing them together to obtain a combined vaccine formulation;
(c) lyophilizing the combined vaccine formulation obtained from the step (b).
According to a preferred embodiment of the present invention, the lyophilization step (c) involves precooling the combined vaccine formulation in the form of aqueous suspension to below the eutectic point temperature of the suspension over 3 to 6 hrs, and then gradually increasing the temperature from xe2x88x9235 to 35xc2x0 C. over 8 to 20 hrs to removing water from the cooled suspension by sublimation.
In a particularly preferred embodiment of the present invention, the prophylactically effective titers of attenuated hepatitis A virus is not less than 106.0 CCID50/ml, and the prophylactically effective, titers of attenuated measles virus is not less than 103.5 CCID50/ml in the lyophilized combined vaccine.
Essentially, according to the present invention, there is provided a combined hepatitis A-measles vaccine consisting of prophylactically effective titers of attenuated live HAV and attenuated live MV, which does not induce any incompatibilities or mutual interference on immune response and immunogenicity between the two viral antigens, and which optionally contains a stabilizer or lyoprotectant.
Measles is the most contagious disease know to man host caused by measles virus (MV). Immunity to measles following an attack of the desease appears to be lifelong. Following measles vaccine, immunity is similarly of many years duration and probably lifelong. The majority of measles cases occur in persons who have not been immunized against measles, and there are small number of people who do not respond to a single dose of measles vaccine. For this reason, since 1989, the recommendation for two does of measles vaccine was made. Therefore, all young children age one year and older should receive a dose of measles vaccine, and the second dose of measles vaccine should be given upon school entry (age 4-6 years) or at 10 to 11 years of age. In addition, persons entering collage, the military, and health profession schools or those who lack a history of childhood illness or immunization should receive a second dose of vaccine. In view of the age shift upward of measles infections, it is particularly important that adolescents returning to school this fall be up-to-date with two doses of measles vaccine.
Hepatitis A (HA) is a worldwide distributive acute communicable discase caused by intection with hepatitis A virus (HAV). Like other enteric deseases mainly transmitted through a faecal/oral route, hepatitis A is typically a childhood infection associated with poor and overcrowded conditions. Athough children hepatitis A is generally very mild, young children play an important role in the transmission of hepatitis A virus because a child with a subclinical infection can pass the virus to non-immune individuals. While improved sanitation and living condition have led to a reduced circution of the virus among some groups of population, younger generations are still susceptible to infection and can act as foci of hepatitis A outbreaks. Vaccination of children attending day-care centers, who live in areas with elevated anti-HAV seroprovalence, has been shown to be of benefit, and it has been suggested that routine hepatitis A vaccination program of children would be the most effective means of controlling HAV infection.
In view of considerable similarity of HAV and MV in epidemiological aspects and a similar two-dose immunization schedule of both hepatitis A and measles vaccine, the desirability and availability of combined hepatitis A-measles vaccine are in evidence.
Up to now, the live measles vaccine generally used in China has been developed from the isolated Chang-47 and Hu-191 strains which have adapted to passage in vitro in primary culture of chick embryo cells. However, studies of the present inventors found that human fetal lung diploid cell monolayers or cell suspension used as cell substrates will advantageous to propagation of the virus and maintenance of viral antigenicity, and also prevented the contamination from exogenous factors, for example egg protein and the like.
On the other hand, the hepatitis A live vaccine has been developed from wide-type HAV strain L-A-1 isolated from stool of a patient by serial passaging in vitro in human diploid fibroblast cells by the precent inventors, and which have been licensed for practical use and industrial-scale production in China. More than 80 milions individuals who are at high risk of hepatitis A exposure received inoculation of the vaccine so far, and the follow-up study results showed a 85% seroprotection rate after primary dose, and a 100% seroprotection rate after the only booster injection.
During their long-term studies and production practies, the present inventors had found that the human fetal lung diploid fibroblast cells (strain 2BS) are exellent host not only for hepatitis A virus, but also for measles virus, and that the measles virus exhibited a more rapidly propagation rates on these cell substrates. In particularly, the present inventors had unexpectedly found that, after inoculating a seed virus of HAV into the diploid cell culture and culturing the cells in a culture medium for 2-3 weeks, an additional inoculation and cultivation of measles virus can be performed, while the infectious potency of HAV achieved a sufficiently high level. After about one additional week, the two viruses could be almost simultaneously harvested in a high recovery and in high intectious potencies. This finding constituted the basis of preparing combined HM vaccine of the invention by propagating the two attenuated live viruses on the same host cell substrate.
In the case of preparing the stock materials of HAV and MV, the respective stock materials of live virus can be diluted and mixed at a suitabe volume ratio, to thereby obtain a combined live vaccine. in the form of aqueous suspension comprising prophylactically effective titers of HAV and MV. Herein, the prophylactically effective titers are refer to a viral potency of not less than 106 CCID50 for HAV and of not less than 103.5 CCID50 for MV.
The present invention further provides a method of preparing combined hepatitis A-measles vaccine, comprising following steps:
(a) providing seed viruses of attenuated hepatitis A live virus and attenuated measles live virus, respectively;
(b) inoculating the seed virus of hepatitis A virus obtained from the step (a) into a human fetal lung diploid fibroblast cell cultures and following by culturing the cells to propagate the virus;
(c) When the positive hepatitis A virus-infected cells are more than 75%, inoculating the seed virus of measles virus obtained from the step (a) into the same cell monolayers on which the hepatitis A virus had propagated, and the cells are cultured continuasly;
(d) At the time when the positive hepatitis A virus-infected cell are more than 90% as detected in terms of immunofluorescence and the positive measles virus-infected cells are more than 90% as detected in terms of cytopatliogenic effect, the cells infected with the two viruses are harvested and the vaccine stock materials containing the two viruses are collected to thereby obtain the desired combined vaccine.
For preparation of the combined HM vaccine, according to the present invention, the vaccine stock material of HAV can be prepared from HAV strain L-A-1 by the method described in CN Patent No.92114998.0, and the vaccine stock material of MV can be prepared from MV strain Chang-47 by a conventional process in accordance with the xe2x80x9cReguirement for Measles Vaccine, livexe2x80x9d in Chinese Reguirements for Bioloical Products.
HAV isolated from clinical materials can grow and propagate in many types of cultured cell culture, though the relative propagation coefficient is lower, and can not lead to a morphologically visual cytopathic changes. By contract, MV can grow and propagate in many types of primary cell culture or subculture in a relatively shoter replication cycle and the typical multinucleate giant cells as well as other similar cyto-morphological changes can be bserved on microscopic examination. The present inventors have showed that human diploid cells are exellent susceptible host for both HAV and MV, so the diploid cell line 2BS is chose as a preferred common host cell used for preparation of the combined HM vaccine of the present invention.
To prepare the combined hepatitis A-measles vaccine, a human diploid cell line 2BS is cultured for growth and maintenance of the cells, and to form a dense confluent cell monolayer in a roller bottle apparatus. Then, a seed virus of HAV is inoculated into the cell culture at a multiplicity of infection (m.o.i.) of 0.02-10 and followed by culturing at about 32-35xc2x0 C. During the culturing of the virus, the area of the infected cells gradually expands in accordance with the propagation of virus and the percentage area of the positive intected cells are monitored by immunofluorescence.
After about 2-3 weeks when more than 75% of HAV-intected cells occurred positive immunofluorescence, change the midium to a nutrient midium containing 10% fetal calf serum (FCS) or 0.01-0.25% (w/v) HSA, and a seed virus of MV strain is additionally inoculated into the same cell culture at a m.o.i. of 0.01 to 10. Then, the human diploid cell culture infected with both HAV and MV is cultured continuasly with renewed the midium at 3-5 days intervals, and examined morphological changes of the cells day by day. The specific morphological changes of the MV-infected cells as the so-called cytopathogenic effect (CPE) and thus the degree of virus growth can be determind. At the time when the CPE is observed almost throughout the entire region of the cell culture monolayer (about 90%) and the positive HAV-infected cells are also more than about 90%, the virus culturing is terminated. Thereafter, the cell culture is placed at 2-8xc2x0 C. for 1-3 days for cold release of the viruses, and then the infected cells can be collected and stored frozen at the temperature of below xe2x88x9220xc2x0 C.
After thawing the cells, the resultant cell suspension is subjected to three cycles of freeze-thawing and subsequently to sonification in a ice water bath for disruption of the cells, followed by centrifugation to remove the cell debris and to separate a supernatant from the suspension. The supernatant containing HAV and MV is collected to obtain a stock material of the combined hepatitis A-measles vaccine.
It is evident that, as mentioned above, the combined HM vaccine can be obtained by a simply mixing process. For the preparation of the mixed vaccine, respective stock material of live vaccines are diluted and mixed so that the dosage levels of each virus maintain a generally fixed antigenic activity employed to elicit an immune response against infection of the viruses.
The present invention still further provides a method of preparing lyophilized combined hepatitis A-measles vaccine, comprising following steps:
(a) providing stock materials containing prophylactically effective titers of attenuated hepatitis A live virus and of attenuated measles live virus respectively, and mixing them to obtain a mixed vaccine stock;
(b) adding stock solution of a stabilizer to the mixed vaccine stock of step (a) at about 1:1 volume ratio and mixing them together to obtain a combined vaccine formulation;
(c) lyophilizing the combined vaccine formulation obtained from the step (b).
In a preferred embodiment, the stock materials of HAV and MV and stabilizer for the lyophilized combined vaccine can be suitable diluted and mixed at about 1:1 (v/v) ratio, and then the resultant aqueous formulation comprising prophylactically effective titers of HAV and MV and stabilizer included in the vaccine formulation at a concentration sufficient to stabilize the viruses againt heat inactivation can be lyophilized, so that the combined HM vaccine thus obtained can be preserved at ambient temperature for a long-term without loss of their infectious potencies, thereby to offer more convenience, potentially better compliance and lower administration costs.
The stabilizer of an attenuated live vaccine can be formulated by a simple mixing each of the components except HSA, and preheating the resultant mixture at about 37xc2x0 C. for 24-48 hrs, then cooling the mixture solution to about 32xc2x0 C. and adding HSA which has been sterilized by a series of ultrafiltration into the mixture solution.
Thereafter, the stabilizer thus obtained can be mixed with virus stock of the combined HM vaccine at a suitable ratio, for example at about 1:1 (v/v) ratio.
However, one of ordinary skill in the art will be well aware that changing volume ratio of stabilizer to vaccine may be applied to practice the claimed invention, which in turn will require changes to the concentration of stabilizer components. The invention not ony limited to the specified 1:1 stabilizer/virus combination to generate the final vaccine formulation for lyophilization. Therefore, the artisan may shose different ratio or use the stock of viral preparation with altered concentration of chemical components which used in preparing stabilizer.
After the vaccine is formulated, the resultant aqueous suspension of the combined vaccine can be dried by lyophilization. Briefly, lyohilization involves the step of precooling the aqueous suspension at below the eutectic point temperature (below xe2x88x9230xc2x0 C.) of the aqueous suspension for about 3-6 hrs, and then removing water from the cooled suspension by sublimation to form a lyophilized vaccine. Within one preferred embodiment, aliquots of the formulated vaccine suspension are subjected a multistep freeze-drying procedure in a refrigerated chamber of the freeze-drying apparatus during a period of 8-20 hrs with gradually uprise of the temperature from about xe2x88x9235xc2x0 C. to 35xc2x0 C.
In order to demonstrate improvement in thermo-stability and storage stability of live vaccine resulted from the presence of stabilizer, the present invention is exemplified by the combined HM vaccine, for example by detecting viral titers pre- and post-lyophilization of the combined HM vaccine, to observe the effectiveness of stabilizer on storage stability of the combined live vaccine. The results show that the stabilizer included in the vaccine formulation of the present invention at a concentration sufficient to stabilize the live virus vaccine against heat inactivation remarkably improved thermal stability of the. vaccine which has been lyophilized as measured by the CCID50, as compared with control vaccine formuation without stabilizer therein.
In a especially preferred embodiment, the stabilizer is essentially composed of from 0 to 2% (w/v) of human serum albumin, from 0.5 to 1% (w/v) of geltion, from 5 to 10% (w/v) of trehalose, from 0.75 to 1.5% (w/v) of sodium glutamate, from 0.05 to 0.55% (w/v) of ascorbic acid, from 0.5 to 2.8% (w/v) of urea, from 5 to 10% (w/v) of mannitol or sorbitol or a 1:1 tixture of them, and from 0.5 to 1% (w/v) of inositol, in terms of the concentrations in the lyophilized live vaccine formulation.
In a comparason experiment, the present inventors found that a similar result can be observed when a stabilizer solution which minus HSA compnent is used in a live virus (especially some envelope-free viruses, for exmaple HAV, MV, and poliovirus) vaccine formulation if the lyophilization cycle parameters could be suitable adjusted.
It can be show experimentally that the combined hepatitis A-measles vaccine of the present invention exhibited a exellent specificity as demonstrated in vitro by serum neutralization (NT) test or virus identification test of MA and HAV. The results indicated that the HAV and MV viruses included in the combined vaccine formulation of the present invention at an adentifiable infectious dose can be completely neutralized by respective specific antisera.
Further, the combined HM vaccine is used to inoculate rhesus monkeys and a series of experiments in viro have done for evidence of its safty and imunogenicity in these higher primates. It have been found that the aminals receved the vaccine are entirely free from paralysis or any other localizing symptoms of central nervous system (CNS), and none of the animals have evidence of histopathologic changes in CNS and hepatic tissue. Egually important, there are no histologic changes that are temporarily related to seroconversion in the infected animals, and also no abnormal elevation of live enzymes which attributable to the inoculum. On the other hand, it has been proved that all of five animals infected with both HAV and MV are developed IgM anti-HAV and IgM anti-MV detectable by enzyme linked immunosarbent assay (ELISA) on weeks 2 to 4 following inoculation, and the serum antibodies are disappeared at weeks eight and nine.
Furthermore, half of animals are developed both specific anti-HAV antibody and anti-MV hemagglutination inhibition (IH) antibody on week two, and the seroconversion rates (sero-prevalences) of both anti-HAV and anti-MV are 100% on week four post-inoculation. Finally, in order to futher demostrate effectiveness and clinical utility of the combined hepatitis A-measles divalent live vaccine, a clinical trial is conducted in which the live hepatitis A vaccine is administered concomitantly with the live measles vaccine, and compared to single administration of monovalent hepatitis A vaccine and monovalent measles vaccine which have been licensed for general distribution in China.
A total of 275 healthy intants aged 8 to 12 months are divided into three groups, and are given one dose of hepatitis A vaccine with measles vaccine, single hepatitis A vaccine and single measles vaccine respectively, for comparing clinical and antibody responses to these three vaccination schemes. During 72 hrs of observation period post-inoculation, none of these subjects occurred both local and general reactions, and there are no significant difference in the rate of adverse events between the groups simultaneous or single administration of HA vaccine and measles vaccine. Especially, between the two groups of receiving either HA vaccine or measles vaccine and combined NM vaccine, no statistically significant differences are observed in the positive seroconversion rates for anti-HAV and anti-MV, and in the geometric mean titers (GMTs) of anti-HAV and anti-MV antibodies, as measured by a modifired ELISA assay.
In summry, both hepatitis A live vaccine and measles live vaccine not only have a similar vaccination schedules for intants and young children, but also without any mutual interference in the immune response and the immunogenicity as demonstrated using identical immunoassay for evoluation. The results as described in Example 4 and 5 of the specification confirmed the outstanding safety and immunogenicity of the combined HM vaccine and support its use in preexposure prophylaxis against the two viruses intection. Therefore, it is expected that the combined hepatitis A-measles vaccine can be prepared from virus stocks of HAV and MV by the methods outlined as above which involves much more techniques than the simple mixing of existing viral antigens, for example co-cultivation of two or more attenuated live viruses on a same cell culture, even though there are many probable problems came from viral genetics and molecular biology remain to be resolved, and would be incorporated into the Children""s Vaccine Initiative for simplifying the implementation, increasing the acceptance and improving the control of the two deseases.
In accordance with the present invention, the combined hepatitis A-measles live vaccine as first dose could be administered to infants aged between 10 to 13 months to provide dual protection against hepatitis A and measles, or as boost dose to young children upon school entry (age 4 to years) or at 10 to 11 years of age. In addition, persons entering college, the military and health profession schools or those lock a history of childhood illness or immunization should receive a second dose of the combined vaccine. In view of age shift upward of measles and hepatitis A infections, it is particularly important that adolescents returning to school with two doses of the combined vaccine.